Competitive survival of clonal serial Pseudomonas aeruginosa isolates from cystic fibrosis airways in human neutrophils

Abstract

Chronic airway infections with Pseudomonas aeruginosa are the major co-morbidity in most people with cystic fibrosis (CF) sustained by neutrophils as the major drivers of lung inflammation, damage, and remodeling. Phagocytosis assays were performed with clonal consortia of longitudinal P. aeruginosa airway isolates collected from people with CF since the onset of lung colonization until patient's death or replacement by another clone. The extra- and intracellular abundance of individual strains was assessed by deep amplicon sequencing of strain-specific single nucleotide variants in the bacterial genome. The varied microevolution of the accessory genome of the P. aeruginosa clones during mild and severe courses of infection corresponded with a differential persistence of clonal progeny in the neutrophil phagosome. By simultaneously exposing the ancestor and its progeny to the same habitat, the study recapitulated the time lapse of the temporal change of the fitness of the clone to survive in neutrophils.

Keywords: Immunology; Microbiology; Respiratory medicine.

Graphical abstract

Figure 1

Concept and flowchart of the competition experiments of serial P. aeruginosa isolates in human neutrophils

Figure 2

Clonal P. aeruginosa CF airway isolates selected for competition experiments Mild (blue) and fatal (red) courses of infection are designated by capital letters. Serial P. aeruginosa isolates chosen for competitive fitness experiments in the presence of neutrophils are marked by green dots (early isolates), yellow triangles (midterm isolates), and gray squares (late isolates), respectively. (A) Trajectory of CF patient’s lung function (FEV1% predicted) during colonization of the first persisting P. aeruginosa clone. (B) Phenotype of the selected P. aeruginosa isolates in quantifiable plate assays.

Figure 3

Characteristics of the human donors’ neutrophils (A) Sorting of freshly isolated viable CD16⁺ neutrophils by flow cytometry. Uninfected cells showed 94 9% vitality after 60 min (see Table S2). (B) Neutrophil surface marker expression of CD184 (black), BLT1 (dark gray), and FPR1 (light gray) in the absence (circles) and presence of P. aeruginosa CF isolates of distinct competitive fitness, i.e., from a severe course (squares) or a mild course (triangles) (cf. Figure 3) immediately before and after 1 h exposure to bacterial inoculum or medium control. (C) Boxplots of the LDH release from neutrophils during phagocytosis assays with mixtures of serial P. aeruginosa isolates from severe (n = 48 assays, red) or mild courses of infection (n = 48, blue) (∗, p < 0.05; ∗∗, p < 0.01, paired Wilcoxon rank-sum test; p < 0.0001, group comparison by Kruskal-Wallis test). (D) Activity of membrane-bound neutrophil elastase assessed with the FRET – sensor NEmo-2. The kinetics of the cleavage of the intramolecular peptide substrate was not significantly different between neutrophils of 3 biological replicates each from the 4 donors. (E) NET formation in neutrophils during phagocytosis assays. The percentage of neutrophils with NETs was quantified in uninfected (white bars) and infected samples (black bars) by confocal immunofluorescence microscopy. The data represent the mean ± SD of the analysis of randomly taken images from four independent competition assays (no. of images from uninfected samples: 0 h, n = 24; 1h, n = 20; 4h, n = 24; no. of images from infected samples: 1h, n = 24; 4h, n = 36). NET formation was significantly higher in infected samples at the 4 h time point (∗∗∗∗, P_corr < 0.0001, two-tailed unpaired Student’s t test) but was not seen at the 1 h time point. (F) Representative overlay images of NET components (blue = DNA, green = DNA/histone-1 complexes, red = myeloperoxidase) of uninfected and infected neutrophils (scale bar = 100 μm). Settings of the microscope were adjusted to the respective isotype control.

Figure 4

NET detection in neutrophils infected with P. aeruginosa clinical isolates of CF patients Shown are the three different categories of NET detection after 60 min and 240 min: nuclear vesicles, cytoplasmic NETs, and NET surface coverage for a mild (course D; white bars) and a severe course (course E; black bars). NETs are marked by arrows. Significance was calculated by Mann-Whitney test; ∗∗∗p < 0.0001. The lower two panels show electron micrographs of nuclear vesicles, cytoplasmic, and NET surface coverage after 240 min exposure to serial P. aeruginosa isolates from a mild (course D; upper panel) or severe course (course E; lower panel). NETs (identified by immunostaining of citH3 and human neutrophil elastase) are marked by arrows. All aspects of NET formation were more strongly induced by serial P. aeruginosa isolates from severe courses than by those from mild courses.

Figure 5

Intra- and extracellular persistence of communities of serial P. aeruginosa CF airway isolates during phagocytosis assays with human neutrophils Proportions of the individual clonal P. aeruginosa isolates of all 12 longitudinal patient courses (A – N) prior to and after 30 s, 30 min, and 60 min co-incubation with neutrophil granulocytes were determined by amplicon sequencing of strain-specific SNVs. The normalized values depict the average of the technical replicates of single phagocytosis assays with the neutrophils of donors 1 and 3. The outcome of the second biological replicate and the phagocytosis assays with the neutrophils of donors 2 and 4 are depicted in Figures S1–S3.

Figure 6

Higher fitness of early P. aeruginosa isolates from severe infections to persist in neutrophils The figure indicates the probability density distribution of the relative change of the proportion of individual P. aeruginosa strains in the intracellular and extracellular compartment during phagocytosis assays at time points 30 s, 30 min, and 60 min. The relative change describes the change of the proportion of individual strains compared to the initial inoculum at a given time point and is depicted in logarithmic scale. The density distributions show how often a certain relative change can be found; for example, a maximum close to 1 means that the bacterial isolates retained their initial proportion. Curves with a larger width of the half maximum reflect a higher variability of the change of the strains’ abundance. The distribution of the late isolates of severe courses (I) is shifted to the left in the direction of a relative decrease, and the distribution of the early isolates (G) shows an opposite shift to a relative increase in comparison to the other isolates. Each curve includes data of all phagocytosis assays with the neutrophils of the four healthy human blood donors. Upper Panel. Probability density distributions of all P. aeruginosa isolates from all (A), the mild (B), and the severe (C) courses of the chronic infections of CF patients’ airways. Middle/lower panel. Probability density distributions of all P. aeruginosa isolates from the mild/severe courses of infections differentiated by colonization time into early (D; G), midterm (E; H), and late (F; I) isolates (cf. Figure 1A).